The present invention relates to nucleotide sequences of the tumor suppressor FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof and antibodies thereto. The present invention relates to the use of nucleotide sequences of FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof and antibodies thereto, as diagnostic and therapeutic reagents for the detection and treatment of cancer. The present invention also relates to therapeutic compositions comprising Fhit proteins, derivatives or analogs thereof, antibodies thereto, nucleic acids encoding the Fhit proteins, derivatives or analogs, and FHIT antisense nucleic acids.
Cancer remains one of the most severe health problems in America, accounting for substantial fatality and health costs in society. Tumorigenesis in humans is a complex process involving activation of oncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell 64:235-248). Tumor suppressor genes in humans have been identified through studies of genetic changes occurring in cancer cells (Ponder, 1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 254:1138-1146).
There is a close association between particular chromosomal abnormalities, e.g., chromosomal translocations, inversions, and deletions, and certain types of malignancy, indicating that such abnormalities may have a causative role in the cancer process. Chromosomal abnormalities may lead to gene fusion resulting in chimeric oncoproteins, such as is observed in the majority of the tumors involving the myeloid lineage. Alternatively, chromosomal abnormalities may lead to deregulation of protooncogenes by their juxtaposition to a regulatory element active in the hematopoietic cells, such as is observed in the translocation occurring in the lymphocytic lineage (Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA 90:9275-9279). Deletions may cause loss of tumor suppressor genes, leading to malignancy.
Nonrandom chromosomal translocations are characteristic of most human hematopoietic malignancies (Haluska et al., 1987, Ann. Rev. Genet. 21:321-345) and may be involved in some solid tumors (Croce, 1987, Cell 49:155-156). In B and T cells, chromosomal translocations and inversions often occur as a consequence of mistakes during the normal process of recombination of the genes for immunoglobulins (Ig) or T-cell receptors (TCR). These rearrangements juxtapose enhancer elements of the Ig or TCR genes to oncogenes whose expression is then deregulated (Croce, 1987, Cell 49:155-156). In the majority of the cases, the rearrangements observed in lymphoid malignancies occur between two different chromosomes.
The TCL-1 locus on chromosome 14 band q32.1 is frequently involved in the chromosomal translocations and inversions with the T-cell receptor genes observed in several post-thymic types of T-cell leukemias and lymphomas, including T-prolymphocytic leukemias (T-PLL) (Brito-Babapulle and Catovsky, 1991, Cancer Genet. Cytogenet. 55:1-9), acute and chronic leukemias associated with the immunodeficiency syndrome ataxia-telangiectasia (AT) (Russo et al., 1988, Cell 53:137-144; Russo et al., 1989, Proc. Natl. Acad. Sci. USA 86:602-606), and adult T-cell leukemia (Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA 90:9275-9279).
In 1979, a large Italian-American family in Boston was observed to be transmitting a constitutional reciprocal t(3;8)(p14.2;q24) chromosome translocation (Cohen et al., 1979, N. Engl. J. Med. 301:592-595; Wang and Perkins, 1984, Cancer Genet. Cytogenet. 11:479-481) which segregated in the family with early onset, bilateral and multifocal clear cell renal carcinoma (RCC). Follow-up cytogenetic studies in several familial tumors demonstrated that the tumors had lost the derivative 8 chromosome carrying the translocated 3p14-pter region; consequently, the tumors were homozygous for all loci telomeric to the 3p14.2 break (Li et al., 1993, Annals of Internal Medicine 118:106-111). It was suggested that the translocation affects expression of a tumor suppressor gene (Cohen et al., 1979, N. Engl. J. Med. 301:592-595) and several investigators have sought candidate suppressor genes. We had suggested the protein tyrosine phosphatase gamma gene (PTPRG) as a candidate tumor suppressor gene (LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040), and that the majority of clear cell RCCs exhibit loss of heterozygosity of a 0.5 Mb region flanking the translocation (Lubinski et al., 1994, Cancer Res. 54:3710-3713; Druck et al., 1995, Cancer Res. 55:5348-5355), although we did not observe aberrations in the remaining PTPRG gene. The 3p14.2 region is also included in deletions in numerous other tumor types, including nasopharyngeal carcinomas (Lo et al., 1994, Int. J. Oncol. 4:1359-1364).
The t(3;8) translocation breakpoint was cloned and a 3 kb transcript of a candidate tumor suppressor gene was detected using a probe from near the breakpoint (Boldog et al., 1993, Proc. Natl. Acad. Sci. USA 90:8509-8513); further details concerning this transcript have not been reported in spite of a later publication from this group relating to this subject, and reporting a YAC contig of approximately 6 Mb DNA spanning the 3p14.2 3;8 translocation breakpoint (Boldog et al., 1994, Genes, Chromosomes and Cancer 11:216-221). It has also been suggested that there may not be a suppressor gene at 3p14.2, that in fact the t(3;8) translocation was a mechanism for losing the von Hippel-Lindau gene, a tumor suppressor gene at 3p25 (Gnarra et al., 1994, Nature Genet. 7:85-90).
Another cytogenetic landmark in chromosome region 3p14.2 is the most common of the constitutive aphidicolin inducible fragile sites, FRA3B, which is cytogenetically indistinguishable from the t(3;8) translocation (Glover et al., 1988, Cancer Genet. Cytogenet. 31:69-73). Fragile sites, of which over 100 have been described in human (for review, see Sutherland, 1991, Genet. Anal. Tech. Appl. 8:1616-166), are regions of the human genome which reveal cytogenetically detectable gaps when exposed to specific reagents or culture conditions; several folate sensitive, heritable, X-linked and autosomal fragile sites have been localized to unstable CCG or CGG repeats (Yu et al., 1991, Science 252:1179-1181; Kremer et al., 1991, Science 252, 1711-1714; Verkerk et al., 1991, Cell 65:905-914; Fu et al., 1991, Cell 67:1047-1058), and for one of these, the FRA11B at 11q23.3, the CCG repeat is within the 5xe2x80x2 untranslated region of the CBL2 gene, a known protooncogene (Jones et al., 1995, Nature 376:145-149). Also this fragile site, FRA11B, is associated with Jacobsen (11q-) syndrome, showing a direct link between a fragile site and in vivo chromosome breakage (Jones et al., 1994, Hum. Mol. Genet. 3:2123-2130). Because the induced fragile sites resemble gaps or breaks in chromosomes, it has frequently been speculated that fragile sites could be sites of chromosomal rearrangement in cancer (Yunis and Soreng, 1984, Science 226:1199-1204). Previously identified fragile sites have also been shown to be hypermethylated (Knight et al., 1993, Cell 74:127-134); thus methylation of a fragile site in a tumor suppressor gene regulatory region might cause loss of transcription of the suppressor gene, serving as one xe2x80x9chitxe2x80x9d in the tumorigenic process, as pointed out previously (Jones et al., 1995, Nature 376:145-149). These authors also suggested that an important contribution of fragile site expression in tumorigenesis might be to increase the incidence of chromosome deletion during tumorigenesis.
The FRA3B region has been delineated by studies of several groups using rodent-human hybrids; hybrid cells retaining human chromosome 3 or 3 and X, on a hamster background, were treated with aphidicolin or 6-thioguanine (to select hybrids which had lost the X chromosome) and subclones selected. Subclones retaining portions of chromosome 3 with apparent breaks in region 3p14-p21 were characterized for loss or retention of specific 3p markers to determine the position of 3p14-21 breaks (LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040, LaForgia et al., 1993, Cancer Res. 53:3118-3124; Paradee et al., 1995, Genomics 27:358-361).
Alterations in oncogenes and tumor suppressor genes in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) have been described, the most frequent target being alterations of p53 (Takahashi et al., 1989, Science 246:491-494; Chiba et al., 1990, Oncogene 5:1603-1610; Mitsudomi et al., 1992, Oncogene 7:171-180) and retinoblastoma (Harbour et al., 1988, Science 241:353-357; Xu et al., 1994, J. Natl. Cancer Inst. 86:695-699) genes and allelic deletions of the short arm of chromosome 3 (3p) (Kok et al., 1987, Nature 330:578-581; Naylor et al., 1987, Nature 329:451-454; Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). In addition to cytogenetically visible deletions (Whang-Peng et al., 1982, Science 215:181-182; Testa et al., 1994, Genes Chrom. Cancer 11:178-194), loss of heterozygosity (LOH) at loci on 3p has been reported in nearly 100% of SCLC (Kok et al., 1987, Nature 330:578-581; Naylor et al., 1987, Nature 329:451-454; Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al., 1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256) and in 50% or more of NSCLC (Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al., 1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256; Rabbitts et al., 1990, Genes Chrom. Cancer 2:231-238; Hibi et al., 1992, Oncogene 7:445-449; Yokoyama et al., 1992, Cancer Res. 52:873-877; Horio et al., 1993, Cancer Res. 53:1-4), strongly suggesting the presence of at least one tumor suppressor gene in this chromosomal region.
However, the observation that allelic losses often involve most of the 3p has hampered the isolation of the involved gene(s). Candidate loci have been identified such as the von-Hippel Lindau gene, located at 3p25, which was subsequently found to be rarely mutated in lung cancer cell lines (Sekido et al., 1994, Oncogene 9:1599-1604). Other loci located in a region within 3p21 were reported to be sites of recurrent homozygous deletions in SCLC (Daly et al., 1993, Oncogene 8:1721-1729; Kok et al., 1993, Proc. Natl. Acad. Sci. USA 90:6071-6075;:Kok et al., 1994, Cancer Res. 54:4183-4187). In addition, transfer of subchromosomal fragments of the region 3p21.3-p21.2 to tumor cell lines has suggested tumor suppressor activity (Killary et al., 1992, Proc. Natl. Acad. Sci. USA 89:10877-10881; Daly et al., 1993, Oncogene 8:1721-1729). More proximal deletions in the 3p12-14 region have also been reported (Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105; Rabbitts et al., 1990, Genes Chrom. Cancer 2:231-238; Daly et al., 1991, Genomics 9:113-119).
Lung cancer is a major cause of mortality worldwide and the overall survival rate has not improved significantly in the last 20 years. Despite the success achieved by primary prevention, lung cancer is still an overwhelming medical and social problem. Even in the cohort of ex-smokers lung cancer incidence remains high for several years, as a consequence of the accumulated damage, and there is an objective need for strategies aimed at reducing cancer mortality in individuals who have stopped smoking.
There remains an unfulfilled need to isolate and characterize the genes associated with digestive tract and other cancers for use as a diagnostic and therapeutic/prophylactic reagent in the detection, treatment, and prevention of such cancers.
Citation of a reference hereinabove shall not be construed as an admission that such reference is prior art to the present invention.
The present invention relates to nucleotide sequences of FHIT genes, and amino acid sequences of their encoded FHIT proteins, as well as derivatives (e.g., fragments) and analogs thereof, and antibodies thereto. The present invention further relates to nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences as well as equivalent nucleic acid sequences encoding a FHIT protein. In a specific embodiment, the FHIT genes and proteins are human genes and proteins.
Mutations (in particular, deletions) of FHIT gene sequences are associated with esophageal, gastric, colon, kidney, and other cancers.
The present invention also relates to expression vectors encoding a FHIT protein, derivative or analog thereof, as well as host cells containing the expression vectors encoding the FHIT protein, derivative or analog thereof. As used herein, xe2x80x9cFHITxe2x80x9d shall be used with reference to the FHIT gene, whereas xe2x80x9cFhitxe2x80x9d shall be used with reference to the protein product of the FHIT gene.
The present invention further relates to the use of nucleotide sequences of FHIT genes and amino acid sequences of their encoded Fhit proteins as diagnostic reagents or in the preparation of diagnostic agents useful in the detection of cancer or precancerous conditions or hyperproliferative disorders, in particular those associated with chromosomal or molecular abnormalities, in particular at 3p14.2, and/or decreased levels of expression, or expression of dysfunctional forms, of the Fhit protein. The invention further relates to the use of nucleotide sequences of FHIT genes and amino acid sequences of their encoded Fhit proteins as therapeutic/prophylactic agents in the treatment/prevention of cancer, in particular, associated with chromosomal or molecular abnormalities at 3p14.2, and/or decreased levels of expression, or expression of dysfunctional forms, of the Fhit protein.
The invention also relates to Fhit derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) Fhit protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with Fhit for binding) to an anti-Fhit antibody], immunogenicity (ability to generate antibody which binds to Fhit), and ability to bind (or compete with Fhit for binding) to a receptor/ligand or substrate for Fhit, and ability to multmerize with Fhit.
The invention further relates to fragments (and derivatives and analogs thereof) of Fhit which comprise one or more domains of a Fhit protein, e.g., the histadine triad, and/or retain the antigenicity of a Fhit protein (i.e., are able to be bound by an anti-Fhit antibody).
The FHIT gene and protein sequences disclosed herein, and antibodies to such protein sequences, may be used in assays to diagnose cancers, e.g., digestive tract and airway tumors, associated with chromosomal or molecular abnormalities at 3p14.2, and/or decreased Fhit protein levels or activity by detecting or measuring a decrease in FHIT wild-type mRNA from a patient sample or by detecting or measuring a decrease in levels or activity of Fhit protein from a patient sample, or by detecting an aberrant Fhit DNA, mRNA, or protein.
The Fhit protein, or derivatives or analogs thereof, disclosed herein, may be used for the production of anti-Fhit antibodies which antibodies may be used diagnostically in immunoassays for the detection or measurement of Fhit protein in a patient sample. Anti-Fhit antibodies may be used, for example, for the diagnostic detection or measurement of Fhit protein in biopsied cells and tissues.
The present invention also relates to therapeutic compositions comprising Fhit proteins, derivatives or analogs thereof, antibodies thereto, and nucleic acids encoding the Fhit proteins, derivatives or analogs, and FHIT antisense nucleic acids.
The present invention also relates to therapeutic and diagnostic methods and compositions based on Fhit proteins and nucleic acids. Therapeutic compounds of the invention include but are not limited to Fhit proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the Fhit proteins, analogs, or derivatives, and FHIT antisense nucleic acids.
The invention provides methods for prevention or treatment of disorders of overproliferation (e.g., cancer and hyperproliferative disorders) by administering compounds that promote Fhit activity (e.g., Fhit, an agonist of Fhit; nucleic acids that encode Fhit).
The invention also provides methods of prevention and treatment of disorders of overproliferation, wherein the patient is hemizygous for a dominant-negative FHIT mutation, by administering compounds that specifically antagonize the FHIT mutant nucleic acid or protein (e.g., antibodies or antisense nucleic acids specific to the mutant).
Animal models, diagnostic methods and screening methods for predisposition to disorders, and methods to identify Fhit agonists and antagonists, are also provided by the invention.
The present invention also relates to methods of production of the Fhit proteins, derivatives and analogs, such as, for example, by recombinant means.
In a particular embodiment of the invention described by way of example in Section 6, a human FHIT sequence is disclosed and shown to be mutated in various cancers.
FIGS. 1A-1B. Organization of the FHIT gene relative to the 3p14.2 FRA3B and translocation sites. A scheme of the normal 3p14.2 region is shown (A) with the chromosomal region (not to scale) represented by the top line with positions of STS markers (position of D3S1234 relative to the gene is not known), the FRA3B represented by the hybrid cl3 break and the t(3;8) translocation break point shown. The dashed portion represents the region involved in the homozygous deletions in tumor cell lines. Three of the YAC clones used in developing the above markers, map and cosmid contig are shown with the cosmid contig below and the distribution of exons in the FHIT transcript shown below the contig. Black and dotted boxes represent coding and noncoding exons, respectively; asterisks indicate exons with start and stop codons. One exon (E5) falls within the defined homozygously deleted region. Exons 1 (E1), 2 (E2) and 3 (E3) fall centromeric to the t(3;8) translocation break and exon 4 (E4) and 6-10 E6-E10) flank the homozygously deleted region on the centromeric and telomeric sides, respectively. Organization of types of aberrant transcripts from tumor cell lines are illustrated in part B, with zigzag regions representing insertions of new sequence, usually repetitive, into the aberrant transcripts. CCL234 and 235 are colon carcinoma-derived cell lines in which homozygous deletion in the fragile region was not detected. In CCL234 RNA, only an abnormal-sized FHIT transcript was detected by RT-PCR amplification and sequencing; the shorter transcript was shown to result from splicing of exon 3 to exon 5, with omission of the noncoding exon 4, leaving the coding region intact. With CCL235 RNA as template, apparently normal and aberrant RT-PCR products were amplified, with the aberrant product resulting from splicing of exon 4 to exon 8 with a repetitive insert of 140 bp (contributing an in frame Met codon) between E4 and E8. RT-PCR amplification of RNA from HeLa cells, a cervical carcinoma-derived cell line which exhibited a deletion or a rearrangement of DNA near the t(3;8) translocation, revealed normal and aberrant-sized products, the smallest product resulting from splicing of exon 4 to exon 9. RT-PCR amplification of RNA from KatoIII, a gastric carcinoma-derived cell line with discontinuous deletions involving the D3S1481 locus and an xcx9c50 kbp region between exons 5 and 6, apparently leaving all FHIT exons intact, resulted in only an aberrant-sized product which is missing exons 4 through 7, with an 86 bp repeat, inserted downstream of exon 3, contributing an in frame Met codon. Amplification of the RT product from HT29, a colon carcinoma-derived cell line with a large deletion (xcx9c200 kbp, about the size of the 648D4 YAC), which included exon 5, gave only an aberrant-sized product resulting from splicing of exon 3 to exon 7. Numerous other tumor-derived cell lines from lung carcinoma (1/3 tested), osteosarcoma (1/1), NPC (3/3), ovarian carcinoma (2/2), and hematopoietic (4/5) tumors, exhibited aberrant FHIT transcription products. The RF48 cell line, from a stomach carcinoma without deletion, showed a normal-sized product, as did a lymphoblastoid line with the t(3;8) translocation, a melanoma (WM1158) and a kidney carcinoma (RC17)-derived cell line. Other colon and stomach carcinoma-derived lines with deletion (AGS, LS180, LoVo), or without deletion (Colo320), showed aberrant reverse transcriptase-polymerase chain reaction (RT-PCR) products (not shown).
FIGS. 2A-2B. Structure of normal and aberrant FHIT cDNAs. The nucleotide (SEQ ID NO:1) and predicted amino acid (SEQ ID NO:2) sequences of the FHIT gene are shown (A) with positions of exons indicated by arrowheads above the sequence and positions of primers used in nested PCR and RACE reactions indicated by arrows below the sequence. A schematic presentation of some of the aberrant transcripts observed in uncultured tumor tissues of digestive organs is shown in B. Only transcripts which showed deletion of coding sequence in Table 3 are presented. The top line in B shows the intact FHIT cDNA map. The thick and thin bars show the coding and untranslated regions, respectively. The positions of splice sites are shown by downward arrows, according to the nucleotide numbers shown above in A. The class I transcripts lack exon 5 while class II transcripts retain exon 5 but generally lose exon 8. In the transcripts with asterisks, insertions of various lengths were observed downstream of exon 4. E1-10 indicate exons 1-10.
FIGS. 3A-3C. Expression of the FHIT gene in normal tissues and tumors. Northern blot (A, B) and RT-PCR analysis (C) of normal and tumor-derived FHIT mRNA. Panel A shows a northern blot of normal mRNAs (2 xcexcg/lane) from spleen (lane 1), thymus (lane 2), prostate (lane 3), testis (lane 4), ovary (lane 5), small intestine (lane 6), colon (mucosal lining) (lane 7), and peripheral blood leukocytes (lane 8), hybridized with the FHIT cDNA probe. Panel B shows a northern blot of mRNAs (2 xcexcg/lane) from normal small intestine (lane 1) and mRNAs from tumor-derived cell lines: KatoIII (lane 2), HK1 (lane 3), LoVo (lane 4), CNE2 (lane 5), CNE1 (lane 6), Colo320 (lane 7), LS180 (lane 8), hybridized with the FHIT cDNA probe (panel B, upper). The same blot was hybridized with a xcex2-actin cDNA probe (panel B, lower). Panel C shows amplified products observed after nested RT-PCR amplification of mRNAs from matched uncultured tumor (T) and normal (N) tissues of the same patients (J4, 9625, 5586, E37, E32, E3), or mRNAs from tumor tissues only (J9, J7, J3, J1, E3). Arrowheads show the positions of amplified products with abnormal DNA sequence. The details of the DNA sequences of corresponding transcripts are shown in Table 2, and FIG. 2B. White dots in the tumor lanes show the position of transcripts with normal DNA sequence.
FIGS. 4A-4B. (A) Alignment of amino acid sequences of HIT family proteins and translation of FHIT cDNAs. Alignment was performed using BOXSHADE version 3.0. Outlining in thick lines indicates two or more identical residues at a position; outlining in thin lines indicates similarity. The PAPH1 (SEQ ID NO:3) (accession #U32615) and CAPH1 (SEQ ID NO:4) (accession #U28374) designate the S. pombe and S. cerevisiae diadenosine 5xe2x80x2,5xe2x80x2xe2x80x3-P1, P4 tetraphosphate asymmetric hydrolases (aph1). FHIT (SEQ ID NO:6) indicates the HIT family member from the cyanobacterian Synechococcus Sp. (accession #P32084), BHIT (SEQ ID NO:5), the protein kinase C inhibitor from B. Taurus (bovine; accession #P16436)), MHIT (SEQ ID NO:7) from M. hyorhinis (mycoplasma, accession #M37339), YHIT (SEQ ID NO:8) from S. cerevisiae (accession #Q04344); the Fhit protein is 69% similar to the S. pombe (PAPH1) gene over a length of 109 amino acids. (B) In vitro translation products from recombinant plasmids containing different alleles of the FHIT gene: pFHIT1 with a deletion of noncoding exon 4 (lane 1); pFHIT2 with an insertion of 72 bp between exons 4 and 5 (lane 2); pFHIT3 with a wildtype FHIT lacking exon 1 (lane 3); the pFHIT full-length wildtype gene in Bluescript (lane 4); control reaction, in vitro translation from the pBCAH vector, carrying a portion of the extracellular region of the PTPRG gene (predicted molecular weight 40 kDa) (lane 5).
FIG. 5. Organization of the FHIT gene relative to documented chromosome breaks in the 3p14.2 fragile region. One FHIT allele is disrupted in all the translocation carriers of the t(3;8) family, with exons 1, 2 and 3 remaining on the derivative 3 chromosome and exons 4-10, including the entire coding region, being translocated to the derivative 8 chromosome, as illustrated above. The hybrid cell line, cl3, with a de novo FRA3B break just telomeric to exon 5, has lost most of the FHIT coding region. The KatoIII cells apparently retain all FHIT exons but encode only an abnormal transcript which lacks exons 4-7 and thus cannot produce Fhit protein. The MB436 and HT29 cells have both lost exon 5 through deletion of different segments of the fragile region.
FIG. 6. Hydrophilicity plot of the Fhit deduced protein sequence (SEQ ID NO:2), plotted using the PEPPLOT program of the Wisconsin GCG software for DNA and protein analysis.
FIGS. 7A-7C. Printout of R50713 nucleotide sequence (SEQ ID NO:9) aligned with the FHIT cDNA sequence (cDNA 7F1) (SEQ ID NO:1), and the R11128 nucleotide sequence (SEQ ID NO:77). The FHIT coding region starts at nucleotide 363 and ends at nucleotide 812.
FIG. 8. Translation in all three reading frames, both 5xe2x80x2 and 3xe2x80x2 directions, of the R50713 EST sequence. 5xe2x80x23xe2x80x2 Frame 1: SEQ ID NOS:10-15 and 76; 5xe2x80x23xe2x80x2 Frame 2: SEQ ID NOS:16-19; 5xe2x80x23xe2x80x2 Frame 3: SEQ ID NOS:20-25; 3xe2x80x25xe2x80x2 Frame 1: SEQ ID NOS:26-31; 3xe2x80x25xe2x80x2 Frame 2: SEQ ID NOS:32-36; 3xe2x80x25xe2x80x2 Frame 3: SEQ ID NOS:37-40.
FIG. 9. Translation in all three reading frames, both 5xe2x80x2 and 3xe2x80x2 directions, of the R11128 EST sequence. 5xe2x80x23xe2x80x2 Frame 1: SEQ ID NOS:41-44; 5xe2x80x23xe2x80x2 Frame 2: SEQ ID NOS:45-48; 5xe2x80x23xe2x80x2 Frame 3: SEQ ID NOS:49-56; 3xe2x80x25xe2x80x2 Frame 1: SEQ ID NOS:57-58; 3xe2x80x25xe2x80x2 Frame 2: SEQ ID NOS:59-64; 3xe2x80x25xe2x80x2 Frame 3: SEQ ID NOS:65-68.
FIGS. 10A-10B. (A) Alignment of yeast (S. pombe) Ap4A hydrolase sequence (U32615) (SEQ ID NO:69) with FHIT cDNA (cDNA 7F1) sequence (SEQ ID NO:1). (B) Result of search for homology stretches between U32615 and cDNA 7F1.
FIGS. 11A-11B. Expression of the FHIT gene in small cell lung cancer (SCLC). (A) Expression of the FHIT gene by nested RT-PCR analysis in SCLC tumors (T) and matched normal (N) tissues. Case 83L indicates a cell line established from the tumor 83T. Sizes of the amplified products are shown at the right. (B) A schematic presentation of the aberrant transcripts of types I and II observed in tumor tissue of SCLCs. The top line shows the intact FHIT cDNA sequence. The thick and thin bars show the coding and untranslated regions, respectively. The positions of splice sites are shown by downward arrows, according to the nucleotide numbers. Type I transcripts lack exons 4 to 6, while type II transcripts lack exons 4 to 8.
FIGS. 12A-12D. Expression of the FHIT gene in small cell lung cancer and sequences of FHIT transcripts. (A) FHIT amplified products observed after nested RT-PCR of mRNA from tumor (T) and normal (N) tissues of case 45 and from tumor (T), normal (N) and cell line (L) samples of case 83. Arrowheads show the sizes of the amplified products. (B-D) Sequences of the type I and II abnormal transcripts observed in SCLCs. Arrows indicate junctions between exons 3 and 4 in the wild-type transcript (WT), between exons 3 and 7 in the abnormal transcripts of type I and between exons 3 and 9 in the abnormal transcripts of type II. WT sequence: SEQ ID NO:78. Type I sequence: SEQ ID NO:79. Type II sequence: SEQ ID NO:80.
FIGS. 13A-13G. Expression of the FHIT gene in non small cell lung cancer (NSCLC) and sequences of FHIT transcripts. (A) Expression of the FHIT gene by nested RT-PCR analysis in NSCLC tumors (T) and paired normal (N) tissues. Arrowheads indicate the amplified abnormal products. (B-G) Sequences of the abnormal transcripts observed in NSCLC cases 2, 3 and 17. Arrows indicate the junctions of exons 4 to 5 in the wild-type products of cases 2 and 17 (2WT, 17WT) and of exon 3 to 4 in the wild type product of case 3 (3WT). 2A shows the junction between exons 4 and 9 in the abnormal product of case 2, 3A shows the junction between exons 3 and 8 in the abnormal product of case 3, and 17A shows the junction between exons 4 and 8 in the abnormal product of case 17. WT sequence: SEQ ID NO:81. 3WT sequence: SEQ ID NO:82. 17WT sequence: SEQ ID NO:83. 2A sequence: SEQ ID NO:84. 3A sequence: SEQ ID NO:85. 17A sequence: SEQ ID NO:86.